Lack of a Role for Cross-Reacting Anti-Thyroid Antibodies in Chronic Idiopathic Urticaria

Transcription

1 ORIGINAL ARTICLE Lack of a Role for Cross-Reacting Thyroid Antibodies in Chronic Idiopathic Urticaria Jonathan D. Mozena 1,2, Adrienne Tiñana 1,2, Julie Negri 1, John W. Steinke 1 and Larry Borish 1 The etiology of chronic idiopathic urticaria (CIU) is attributed to autoantibodies directed against the a-chain of the high-affinity IgE receptor (FceRIa) or IgE on mast cells in 30 60% of patients. Approximately 30% of CIU patients have Hashimoto s thyroiditis (HT). We investigated the pathophysiologic relationship of anti-thyroid and anti-fceria antibodies. Nine individuals with both CIU and HT underwent autologous serum skin testing (ASST) and sera were assayed for thyroid autoantibodies, thyroid-stimulating hormone, and anti-fceria antibodies. Serum samples were studied for their ability to activate a human mast cell line (LUVA) as determined by cysteinyl leukotriene (CysLT) production. Experiments were performed to determine whether epitope crossreactivity could explain the high incidence of HT found in CIU patients. A significant proportion of CIU patients had a positive ASST (nine of six) and anti-fceria antibodies (six of nine). Incubation of patient sera with FceRIa, but not thyroglobulin or thyroid peroxidase, resulted in the decreased ability to detect anti-fceria antibodies. Incubation with thyroid antigens did not inhibit CysLT production by mast cells. Epitopic cross-reactivity does not explain the increased prevalence of HT found in CIU patients. The frequent concurrence of HT and CIU likely reflects a genetic tendency toward autoimmune diseases. Journal of Investigative Dermatology (2010) 130, ; doi: /jid ; published online 25 February 2010 INTRODUCTION Chronic idiopathic urticaria (CIU) is defined as recurrent episodes of hives with erythema and pruritus occurring over a period of 6 weeks or longer (Sabroe et al., 1999). CIU is a common disorder with a prevalence of 0.1% in the general population (Charlesworth, 2002). In the 1980s, Grattan et al. (1986) reported the presence of a serum factor that induced a wheal and flare response following intradermal injection of autologous serum, thought to be reflective of the presence of autoantibodies that are capable of inducing mast cell (and basophil) degranulation. The autologous serum skin test (ASST) has since been regarded as a reliable in vivo test in predicting autoimmunity in CIU patients (Sabroe et al., 1999). Studies have demonstrated that 30 60% of CIU patients possess IgG autoantibodies directed against the a-subunit of the high-affinity IgE receptor (FceRIa) on mast cells and basophils (Hide et al., 1993; Fiebiger et al., 1995; Ferrer et al., 1998). An additional 5 10% of CIU patients reportedly possess autoantibodies directed against IgE (Gruber et al., 1988). Previous studies 1 Asthma and Allergic Disease Center, Carter Center for Immunology Research, University of Virginia, Charlottesville, Virginia, USA 2 These authors contributed equally to this work. Correspondence: Larry Borish, Asthma and Allergic Disease Center, University of Virginia, PO Box , Charlottesville, Virginia , USA. lb4m@virginia.edu Abbreviations: ASST, autologous serum skin test; CIU, chronic idiopathic urticaria; CysLT, cysteinyl leukotriene; FceRIa, a-subunit of the high-affinity IgE receptor; HT, Hashimoto s thyroiditis; TA, thyroid autoantibodies; TG, thyroglobulin; TPO, thyroid peroxidase Received 14 July 2008; revised 12 January 2010; accepted 13 January 2010; published online 25 February 2010 have demonstrated that IgG from patients with CIU stimulates histamine release from donor basophils in vitro suggesting a pathogenic role for autoantibodies (Grattan et al., 1991). Basophil activation tests have been considered the benchmark diagnostic test to identify autoimmunity as the cause of hives in patients with CIU (Yasnowsky et al., 2006; Greaves and Tan, 2007; Altrich et al., 2009). However, the main limitation to this approach is the inherent variability of basophils, which requires rigorous screening of basophils from numerous donors both to identify releaser basophils capable of FceRI-dependent activation (Lichtenstein and MacGlashan, 1986) and also to ensure that donor basophils do not spontaneously secrete high basal levels of histamine. In addition, although basophils in vitro provide useful diagnostic information, CIU is primarily a disease of cutaneous mast cells. As basophils are not the primary cellular target of CIU, a mast cell model is the more ideal framework to study CIU pathophysiology. We have developed an immortalized human mast cell line (LUVA) displaying high concentrations of FceRI (Steinke et al., 2009). These cells are useful in the study of immune mechanisms of mast cell-dependant disorders such as CIU, insofar as they degranulate and release arachidonic acid-metabolites in an FceRI-dependant manner. The LUVA cells are activated when incubated with CIU serum, and this activation correlates with the presence of a positive ASST (Tiñana A, Mozena J, Steinke J Screening for autoimmune mechanisms in chronic idiopathic urticaria (CIU) utilizing an immortalized human mast cell line (LUVA), abstract submitted to the American Academy of Allergy, Asthma and Immunology annual meeting, February 2010). Thyroid autoantibodies (TA) have a prevalence of B3 6% in the general population versus B15 30% in CIU 1860 Journal of Investigative Dermatology (2010), Volume 130 & 2010 The Society for Investigative Dermatology

2 patients (Leznoff and Sussman, 1989; Turktas et al., 1997; Verneuil et al., 2004; Palma-Carlos and Palma-Carlos, 2005; Cebeci et al., 2006). Although these studies establish the coincidental occurrence of TA and CIU, no current data exist to determine the basis for this linkage. The confluence of these two diseases could reflect an underlying genetic predisposition for the development of autoimmune illnesses. Alternatively, however, it is plausible that these diseases are pathogenically linked, possibly reflecting a shared epitope targeted by these different autoantibodies. Elucidation of the role that TA have in CIU may ultimately help in understanding the pathologic basis of this condition. We initiated the current studies to further study the relationship between the presence of anti-fceria antibodies and TA in patients with CIU. Specifically, we investigated whether the high incidence of Hashimoto s thyroiditis (HT), defined as the presence of antibodies to thyroglobulin (TG) and/or thyroid peroxidase (TPO), observed in patients with CIU could be explained by epitope cross-reactivity of anti- FceRIa antibodies with thyroid antigens. RESULTS Study population Twenty patients with CIU were enrolled in the study. The characteristics of patients enrolled into the study are presented in Table 1 and summarized in Table 2. The majority of patients with CIU and HT were females (17 females/3 males), with an average age of 38±13 years. ASST and anti-fceria antibodies Thirteen of 20 subjects had a positive ASST. In addition, 12 of 20 patients had positive anti-fceria antibodies. Nine of 20 patients had both a positive ASST and anti-fceria antibodies (Tables 1 and 2). Thyroiditis prevalence Nine of 20 subjects were selected for further study based on screening for the presence of either anti-tg or anti-tpo antibodies. Six of nine of these study patients required thyroid replacement therapy, the remainder had euthyroid HT (Table 1). Epitope sharing between FceRIa and thyroid proteins Initially, a CIU patient who possessed anti-fceria, anti-tpo, and anti-tg antibodies was evaluated for cross-reactivity regarding epitope binding. The ability to detect serum anti-fceria antibodies was inhibited in a dose-dependent manner with preincubation of the serum sample with increasing concentrations of soluble FceRIa. However, neither recombinant TPO nor purified TG competed with binding to FceRIa (Figure 1). Subsequently, four additional subjects with anti-fceria, anti-tpo, and anti-tg antibodies were studied for epitope binding cross-reactivity using high concentrations of FceRIa, TPO, and TG. The detection of anti-fceria antibodies by ELISA was inhibited by an average of 93±11% when these patient sera were incubated with human recombinant FceRIa (Table 3). However, the ability to detect anti-fceria antibodies was not inhibited when sera from the same patients were incubated with TPO or TG (Table 3). Functional epitope cross-reactivity As previously reported, serum obtained from CIU patients induced mast cell activation as demonstrated as cysteinyl leukotriene (CysLT) secretion (Tiñana et al., 2009). However, in CIU patients who possessed anti-fceria, anti-tpo, and anti-tg antibodies, CysLT secretion by mast cells was not affected by preincubation with TPO or TG (Figure 2). Functional antibody-mediated mast cell activation As expected, incubation with anti-fceria induced significant CysLT production. In contrast to TPO and TG, this effect was inhibited by preincubation with recombinant FceRIa (490% inhibition) (data not shown). However, incubation with either anti-tg or anti-tpo induced mast cell activation. In addition, incubation with TPO and TG did not interfere with activation of mast cells with anti-fceria (Figure 2). DISCUSSION Consistent with many other autoimmune diseases and similar to what other investigators have reported (O Donnell et al., 2005; Kulthanan et al., 2006), we found that CIU typically affects females in the third or fourth decade of life (Table 1). In support of an autoimmune etiology to CIU, an increased prevalence of HT in patients with CIU is well recognized with a reported range of B15 30% (Leznoff and Sussman, 1989; Turktas et al., 1997; Verneuil et al., 2004; Palma-Carlos and Palma-Carlos, 2005; Cebeci et al., 2006). In contrast, the prevalence of TA in healthy control populations is reported to be %. In our CIU population (Table 1), a slightly higher prevalence of TA was noted (45%). We confirmed the use of the ASST in demonstrating the presence of mast cell-activating function in the serum of CIU patients (Hide et al., 1993; Toubi et al., 2004; Yasnowsky et al., 2006). However, a positive ASST was equally prevalent in patients who did not have TA (67 vs 64%). We also confirmed a high prevalence of anti-fceria antibodies in the serum of CIU patients as detected by ELISA (60%). However, similar to the ASST results, an equivalent proportion of CIU patients with and without TA had positive anti-fceria antibodies (67 vs 55%, respectively). Therefore, the presence of TA does not predict the presence of an autoimmune signature, specifically the presence of a positive ASST or anti-fceria antibodies. The possibility that a larger sample size could detect a CIU/HT autoantibody profile that correlated with the presence of anti-fceria antibodies cannot be discounted. The mechanism by which HT is linked to CIU has not been elucidated. Concha et al. (2004) demonstrated that 2 of 20 patients with CIU and HT possessed measurable IgE antithyroid antibodies, although this has not been confirmed (Tedeschi et al., 2001). We addressed the possibility that epitope binding cross-reactivity could explain the concomitant occurrence of these two autoimmune conditions. However, this was comprehensively ruled out. Neither anti- TG nor anti-tpo antibodies are capable of inducing activation of connective tissue mast cells (Figure 2). In our patients with CIU who possessed anti-fceria, TPO, and TG antibodies, the detection of anti-fceria antibodies by ELISA

3 Table 1. Characteristics of CIU population Patient Sex Age Thyroid replacement TPO antibodies TG antibodies ASST 1 FceRIa antibodies 2 Thyroiditis cohort 1 F 56 No No Yes Negative Positive 2 F 28 No No Yes Positive Positive 3 F 45 Yes Yes No Positive Positive 4 M 65 Yes No Yes Positive Positive 5 F 40 Yes Yes Yes Positive Positive 6 F 29 Yes Yes Yes Negative Negative 7 F 61 Yes Yes Yes Negative Negative 8 F 21 Yes Yes Yes Positive Negative 9 F 24 No No Yes Positive Positive Non-thyroiditis cohort 10 F 32 Positive Negative 11 F 35 Positive Positive 12 F 61 Positive Positive 13 M 29 Negative Negative 14 F 27 Positive Positive 15 M 47 Negative Positive 16 F 46 Positive Negative 17 F 27 Positive Positive 18 F 37 Negative Negative 19 F 33 Negative Positive 20 F 25 Positive Negative Abbreviations: ASST, autologous serum skin testing; CIU, chronic idiopathic urticaria; F, female; FceRIa, a-chain of the high-affinity IgE receptor; M, male; TG, thyroglobulin; TPO, thyroid peroxidase. 1 Positive ASST defined as wheal 45 mm in diameter more than saline controls. 2 Positive anti-fceria titer defined as 43.9 mgml 1. Table 2. Summary of characteristics of CIU population Sex (# female) Age (mean years±sd) ASST (# positive) FceRIa antibodies (# positive) Thyroiditis cohort (n=9) 8/9 41±16 6/9 6/9 Non-thyroiditis cohort (n=11) 9/11 36±10 7/11 6/11 Abbreviations: ASST, autologous serum skin testing; CIU, chronic idiopathic urticaria; FceRIa, a-chain of the high-affinity IgE receptor. was not inhibited by the addition of TG or TPO (Figure 1 and Table 3). Finally, when we investigated functional antibody cross-reactivity, we demonstrated that preincubation with these thyroid proteins did not decrease the capability of CIU serum to activate mast cells (Figure 2). It should be noted that our diagnostic assays for the presence of anti-fceria antibodies might exaggerate their frequency. The recombinant FceRIa we used was produced in a baculovirus cell line that has the machinery to decorate proteins with N-linked glycans thought to have a role in protein folding. Many humans possess pre-existing antibodies to this host cell line-specific glycosylation motif (Hancock et al., 2007). As such, some of the CIU patients in whom we report anti-fceria antibodies may, in fact, only have antibodies to this glycosylation motif. However, our studies (Figure 1) do demonstrate that this glycosylation motif does not interfere with the ability of soluble recombinant FceRIa to capture IgE receptor-targeted antibodies. This latter observation validates our reporting the absence of cross-reacting functional antibodies. In summary, our data confirm the high prevalence of TA and anti-fceria antibodies in CIU. However, this phenomenon cannot be explained by the presence of a shared epitope recognized by these two different antibodies Journal of Investigative Dermatology (2010), Volume 130

4 An alternative explanation for the increased incidence of HT in CIU is that these conditions reflect a shared genetic predisposition toward the development of autoimmune disease. Along the same lines, autoimmune thyroid disease also has a tendency to occur concurrently with type 1 diabetes. This co-association has frequently been found to occur with patients who express HLA-DR3, suggesting that their etiology may involve common genetic factors (Levin et al., 2004). This possibly reflects shared thymic-programmed mechanisms for developing self-tolerance. In summary, our data confirm the high prevalence of TA and anti-fceria antibodies in CIU and strongly suggests that anti-thyroid and anti-fceria antibodies do not share epitope specificity. MATERIALS AND METHODS Patient enrollment Patients with CIU were recruited from the outpatient adult allergy/ immunology clinics at the University of Virginia. Subjects gave written informed consent under a protocol approved by the University of Virginia human institutional review board. The study was carried out in accordance with the Declaration of Helsinki principles. Subjects were diagnosed with CIU based on the presence of urticarial lesions of 46 weeks duration who had hives present at least 3 days per week. All patients had their blood screened for thyroid-stimulating hormone, free T 4 (if thyroid-stimulating hormone abnormal), anti-tpo antibody, and anti-tg antibody. Patients with significant elevation in circulating antibodies to either FcεRlα amtibodies detected (μg ml 1 ) [FcεRIα] Serum alone [TPO] [TG] Figure 1. Inhibition assay using recombinant FceRIa, TPO, and thyroglobulin. Serum from one patient who had anti-fceria and TA was diluted 1:50 in PBS containing 0.05% sodium azide and incubated overnight at 37 1C with increasing concentrations of recombinant human FceRIa, purified human TG, or recombinant human TPO. Titers of anti-fceria antibodies were measured using the methodology described. TPO (40.7 IU ml 1 )ortg(40.6 IU ml 1 ) were defined as having HT and invited to participate in further studies. Patients with a history of autoimmune disease (other than HT), chronic infection, or malignancy were excluded. Antihistamines were discontinued for a minimum of 5 days before skin testing. Patients on immunosuppressive medications (for example cyclosporine, plaquenil) were excluded, as were patients who had used corticosteroids within the previous 2 months. FceRIa antibody assay Phosphate-buffered saline (PBS) with or without 200 ng l 1 of recombinant human FceRIa (Heska, Fort Collins, CO) were incubated overnight and adsorbed onto sterile flat-bottomed Immulon 2HB 96-well ELISA plates (Beckman Coulter, Fullerton, CA). The concentration of 200 ng l 1 FceRIa was determined experimentally Cysteinyl leukotriene production(pg ml 1 ) a b c PBS Alone TG TPO FcεRlα FcεRlα +TG FcεRlα +TPO Serum +PBS Serum +TG Serum +TPO Figure 2. FceRIa functional assay. Comparison of CysLT production after: (a) activation with PBS, anti-thyroglobulin, and anti-tpo antibodies; (b) preincubation with thyroid antigens followed by activation with anti-fceria; and(c) preincubation with thyroid antigens followed by activation with patient serum. (a ) LUVA mast cells were activated with PBS, anti-tg, or anti-tpo antibodies to evaluate if anti-thyroid antibodies caused mast cell activation. (b) Additional mast cells were preincubated with recombinant TPO or purified TG followed by activation with anti-fc eria to determine if thyroid antigens interfere with activation of cells. (c) Sera from patients with positive anti-tg (n ¼ 8) or anti-tpo (n ¼ 5) antibodies were diluted 1:20 and incubated with thyroid antigens overnight. This was followed by activation of LUVA mast cells to determine if preincubation with the thyroid antigens inhibited CysLT production. For all experiments, activation took place over 30 minutes at 37 1 C. Supernatants were collected and CysLT production quantified by ELISA as described. Results presented in panels a and b were from a single experiment with the LUVA cells and thus have no error bars, whereas in panel c the error bars represent the standard error of the mean. Table 3. FceRIa inhibition assay with FceRIa, thyroid peroxidase, and thyroglobulin Serum incubated with recombinant FceRIa Serum incubated with purified thyroglobulin Serum incubated with recombinant thyroid peroxidase % Inhibition of anti-fceria antibody detection (±SD) 93±11* 17±16 8±12 FceRIa, a-chain of the high-affinity IgE receptor. *Po

5 and found to yield comparable results to higher concentrations. The next day, wells were washed and blocked with 10% goat serum in PBS (ph 7.4; Sigma, St Louis, MO) for 4 hours. After washing, plates were incubated with 100 ml human sera in duplicate diluted 1:100 in PBS containing 5% goat serum at 4 1C for 1 hour. Plates were rinsed before adding biotinylated anti-human IgG 1:10,000 (Rockland Immunochemicals, Gilbertsville, PA) in 5% goat serum and incubated for 1 hour at 4 1C. After washing, 100 ml of horseradish peroxidase-conjugated streptavidin (Sigma) diluted 1:10,000 in PBS was incubated in wells for 20 minutes. The ELISA was developed with o-phenylenediamine dihydrochloride 0.4 mg ml 1 (Sigma) in 0.05% phosphate-citrate buffer with sodium perborate (Sigma) for B20 minutes before the reaction was terminated with 3 N sulfuric acid (after the optical density of the most concentrated portion of the standard curve was between 1.5 and 2.0 (see below)). Measured values were translated into antibody concentrations by logarithmic regression analysis using standardized human IgG (Sigma) directly adsorbed to the plate (comprising a linear range from 0 to 1,000 ng ml 1 ). On the basis of preliminary testing performed in a large cohort of CIU patients (data not shown), a positive anti-fceria assay was defined as a concentration of 43.9 mgml 1. Autologous serum skin test ASST was performed using standard methodology (Sabroe et al., 1999). Blood was drawn into a glass tube, allowed to clot, and centrifuged at 5,000 r.p.m. for 15 minutes to separate sera. Patient sera (50 ml) was injected intradermally into the ventral forearm and compared with saline and histamine controls. Skin tests were read after 30 minutes with wheals measuring 45 mm in diameter more than saline defined as positive. FceRIa inhibition binding assay Inhibition assays were performed to determine whether a shared epitope recognized by both anti-fceria and TA could explain the high incidence of HT associated with CIU. Initially one serum sample from a subject who possessed anti-fceria, anti-tg, and anti- TPO antibodies was diluted 1:50 in PBS and incubated overnight at 37 1C with either PBS, recombinant human FceRIa, human TG (Advanced Immunochemicals, Long Beach, CA), or recombinant human TPO (Research Diagnostics, Flanders, NJ) at a range of concentrations and then assayed for the presence of anti-fceria antibodies by ELISA as described. An experiment was then performed with four representative patients who also had all three classes of autoantibodies with high concentrations of recombinant human FceRIa, TG, or TPO (at 65 mgml 1 each). FceRIa inhibition functional assay Serum was diluted 1:20 in PBS and incubated with PBS, recombinant TPO, or purified TG (65 mgml 1 each) overnight as described for the binding assay. The next day, the serum samples were incubated with LUVA mast cells at 37 1C for 30 minutes and supernatants collected. LUVA activation was determined as CysLT secretion as quantified by ELISA according to the manufacturer s instructions (Cayman Chemical, Ann Arbor, MI). FceRIa functional assay LUVA mast cells were incubated with PBS, and commercially available anti-tg (Sigma) and anti-tpo (Biodesign International, Saco, ME) antibodies for 30 minutes at 37 1C and supernatants were collected. Similarly, LUVA mast cells were incubated with recombinant anti-fceria (Upstate, Lake Placid, NY) alone and anti-fceria after preincubating with recombinant soluble FceRIa (Heska) for 15 minutes. In addition, LUVA mast cells were preincubated with recombinant TPO and purified TG for 15 minutes followed by incubation with anti-fceria for 15 minutes. LUVA activation was determined as CysLT secretion as described above. CONFLICTS OF INTEREST The authors state no conflict of interest. ACKNOWLEDGMENTS We thank Dr Donald Wassom of Heska Corporation for providing purified recombinant human FceRIa. We also thank Brandon Early BS, Lucy Goddard RN, and Elizabeth Kropf BS for their technical assistance. This study was supported by a grant from the National Institutes of Health grant AI REFERENCES Altrich ML, Halsey JF, Altman L (2009) Comparison of the in vivo autologous skin test with in vitro diagnostic tests for diagnosis of chronic autoimmune urticaria. Allergy Asthma Proc 30:28 34 Cebeci F, Tanrikut A, Topcu E et al. (2006) Association between chronic urticaria and thyroid autoimmunity. Eur J Immunol 16:402 5 Charlesworth EN (2002) Urticaria and angioedema. Allergy Asthma Proc 23:341 5 Concha LB, Chang CC, Szema AM et al. (2004) IgE antithyroid antibodies in patients with Hashimoto s disease and chronic urticaria. Allergy Asthma Proc 25:293 6 Ferrer M, Kinet JP, Kaplan AP (1998) Comparative studies of functional and binding assays for IgG anti-fceria (a-subunit) in chronic urticaria. J Allergy Clin Immunol 101:672 6 Fiebiger E, Maurer D, Holub H et al. (1995) Serum IgG auto-antibodies directed against the a chain of FceRI: A selective marker and pathogenetic factor for a distinct subset of chronic urticaria patients? J Clin Invest 96: Grattan CE, Francis DM, Hide M et al. (1991) Detection of circulating histamine releasing auto-antibodies with functional properties of anti-ige in chronic urticaria. Clin Exp Allergy 21:695 Grattan CE, Wallington TB, Warin RP et al. (1986) A serological mediator in chronic idiopathic urticaria: a clinical immunological and histological evaluation. Br J Dermatol 114: Greaves MW, Tan KT (2007) Chronic urticaria: recent advances. Clin Rev Allergy Immunol 33: Gruber BL, Baeza M, Marchese M et al. (1988) Prevalence and functional role of anti-ige auto-antibodies in urticarial syndromes. J Invest Dermatol 90:213 7 Hancock K, Someet Narang S, Pattabhi S et al. (2007) False positive reactivity of recombinant diagnostic glycoproteins produced in High Five TM insect cells: effect of glycosylation. J Immunol Methods 330:130 6 Hide M, Francis DM, Grattan CE et al. (1993) Auto-antibodies against the high affinity IgE receptor as a cause of histamine release in chronic urticaria. N Eng J Med 328: Kulthanan K, Jiamton S, Gorvanich T et al. (2006) Autologous serum skin test in chronic idiopathic urticaria: prevalence, correlation and clinical implications. Asian Pac J Allergy Immunol 24:201 6 Levin L, Ban Y, Concepcion E et al. (2004) Analysis of HLA genes in families with autoimmune diabetes and thyroiditis. Hum Immunol 65:640 7 Leznoff A, Sussman A (1989) Syndrome of idiopathic chronic urticria and angioedema with thyroid autoimmunity: a study of 90 patients. J Allergy Clin Immunol 84: Journal of Investigative Dermatology (2010), Volume 130

6 Lichtenstein LM, MacGlashan DW (1986) The concept of basophil releasability. J Allergy Clin Immunol 77:291 4 O Donnell BF, Francis DM, Swana GT et al. (2005) Thyroid autoimmunity in chronic urticaria. Br J Dermatol 153:331 5 Palma-Carlos AG, Palma-Carlos ML (2005) Chronic urticaria and thyroid auto-immunity. Allerg Immunol (Paris) 37:143 6 Sabroe RA, Grattan CE, Francis DM et al. (1999) The autologous serum skin test: a screening test for auto-antibodies in chronic idiopathic urticaria. Br J Dermatol 140: Steinke J, Borish L, Tiñana A et al. (2009) Development of an immortalized stem cell factor (SCF)-independent mast cell line displaying high concentrations of FceRI. J Allergy Clin Immunol 123:S213 Tedeschi A, Lorini M, Asero R (2001) thyroid peroxidase IgE in patients with chronic urticaria. J Allergy Clin Immunol 108:467 8 Toubi E, Kessel A, Avshovich N et al. (2004) Clinical and laboratory parameters in predicting chronic urticaria duration: a prospective study of 139 patients. Allergy 59: Turktas I, Gokcora N, Demirsoy S et al. (1997) The association of chronic urticaria and angioedema with autoimmune thyroiditis. Int J Dermatol 36: Verneuil L, Leconte C, Ballet JJ et al. (2004) Association between chronic urticaria and thyroid autoimmunity: a prospective study involving 99 patients. Dermatology 208: Yasnowsky KM, Dreskin SC, Efaw B et al. (2006) Chronic urticaria sera increase basophil CD203c expression. J Allergy Clin Immunol 117: